Understanding polycyclic aromatic hydrocarbon degradation by soil microbial communites

90 Pages·2009·3.07 MB·English

by  Inigo Walker Howlett

UNDERSTANDING POLYCYCLIC AROMATIC HYDROCARBON
DEGRADATION
BY SOIL MICROBIAL COMMUNITIES:
THE CONCENTRATION AND DISTRIBUTION OF
AROMATIC DIOXYGENASE GENES

A Thesis
Presented for the
Master of Science
Degree
The University of Tennessee, Chattanooga

Inigo Walker Howlett
April 2009
Copyright © 2009 by Inigo Walker Howlett

All Rights Reserved

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ACKNOWLEDGMENTS

I would like to thank my committee, Dr. Joey Shaw, Dr. Margaret Kovach, and my
advisor, Dr. Henry Spratt, for all of their efforts on my behalf. To Dr. Ying Tao, who
unexpectedly came to my assistance with all things molecular, and to Dr. Jennifer M.
Debruyn of the University of Tennessee, Knoxville, for advice and assistance with Real-
Time PCR. To Craig Zeller of the U.S. EPA, and Mike Pressler of the University of
Central Michigan, and to Andy Carrol for all of his help with the GIS aspects of this
work.

Especially, to my family:
To my Grandparents, Maj. William George and Orva Heissenbuttel, for my education,
and to my parents, Iris Heissenbuttel and F. Carey Howlett, and my brother Owen, for
their years of support.

Inigo Walker Howlett

Spring 2009

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ABSTRACT

The ability to individually identify and enumerate bacterial genes in the environment
is an important development in the field of microbial ecology. Researchers are now
able to quantify and map populations and communities, the presence of which could
only be inferred until recently. Knowledge of this ecology is critical to understanding
microbial responses to environmental pollutants, thereby contributing information
useful in devising and monitoring bioremediation strategies. The following thesis
therefore compares two aromatic dioxygenase genes, nidA and nahAc, which confer
the ability for the bacteria carrying the genes to metabolize polycyclic aromatic
hydrocarbons (PAHs), persistent pollutant byproducts of coal coking. The deposition
of PAHs influences the development of soil microbiological communities, creating
selective pressure for the development of microbial communities able to survive. To
confirm that the mineralization of PAHs correlates with gene copy number, 14C
radiorespireometery was used to detect the mineralization of radiolabled
phenanthrene. Due to culture bias, the naphthalene-targeting Pseudomonas proxy
gene nahAc was the first discovered and the longest studied. The degradation of
larger, more persistent PAHs has been found to rely on the Mycobacterium- specific
gene nidA. Soil sampling was conducted at three locations in the Chattanooga region
during the summer of 2007. The samples were analyzed using quantitative real-time
PCR, and the distribution patterns of nidA and nahAc in copies/gDry/gCarbon were
mapped and compared. Differences in gene distribution and concentration were
examined between genes as a broad survey, and between sites to examine response to
PAH exposure. Prior exposure, indicated by active14C phenanthrene mineralization,
was determined by radiorespireometry. The nidA gene was locally predominant over
nahAc (p<0.01), and was detected at every sample site, while nahAc was much more
variable in concentration, with no detection in 17 of 22 samples. The nidA gene
showed an elevated response to prior exposure, occurring at higher levels in the
Chattanooga Creek Superfund site than in sites with no history of industrial activity
(p<0.05). Additionally, nidA copy number had a weakly positive correlation with the
mineralization of 14C phenanthrene (r=0.5594, p<0.1). This establishes nidA as a
more reliable indicator than nahAc of the presence of dioxygenase activities under
current soil conditions. Mapping the concentration and distribution of a reliable and
responsive member of the soil community using GIS technology provides a concise
illustration of the current distribution of targeted genes and their response to PAH
contamination. Advances in the understanding of microbial ecology underlying
contaminated sites may lead to faster, more accurately targeted remediation strategies.

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TABLE OF CONTENTS

INTRODUCTION…………………………………………………… 1

PART I: LITERATURE REVIEW…............................................... 5
MOLECULAR BIOLOGY AND MICROBIAL ECOLOGY 5
HISTORY …......................................................................... 5
BACTERIA AND ENZYMES ………………………………..... 6
GENES AND THEIR BACTERIA…...........................…………. 11
MICROBIAL FACTORS INFLUENCING GENE COPY NUMBER... 11
GEOCHEMISTRY 12
PAH CONTAMINATION……………………………………. 13
DENSE NON AQUEOUS PHASE LIQUID DIFFUSION IN SOIL … 14
SOILS, PH AND BACTERIA………………………..... 16
GEOGRAPHIC INFORMATION SYSTEMS 17

PART II: A SURVEY OF AROMATIC DIOXYGENASE GENES
IN THE CHATTANOOGA AREA………………...... 19
INTRODUCTION...................................................................... 19
BACTERIA CONTAINING NIDA AND NAHAC…………………. 20
CHEMOORGANIC CARBON LOADING……………………….. 21
SEASONALITY........................................................................ 22
MATERIALS AND METHODS………………………………..... 23
SAMPLING LOCATIONS AND METHODS…………………….. 23
DNA EXTRACTION AND QUANTITATIVE REAL-TIME PCR…... 26
ELEMENTAL ANALYSIS........................................................... 27
GIS ANALYSIS……………………………………………… 27
RESULTS …………………………………………………….. 28
DNA EXTRACTION AND QUANTIFICATION………………….. 28
QUANTIFICATION AND MAPPING WITHIN WILLIAMS ISLAND… 30
QUANTIFICATION AND MAPPING WITHIN VOLUNTEER ARMY
AMMUNITION PLANT (VAAP)………………………………. 32
QUANTIFICATION AND MAPPING WITHIN CHATTANOOGA
CREEK…………………………………………………. 33
STATISTICAL COMPARISON OF QUANTIFICATION DATA …….. 35
DISCUSSION …………………………………………………. 37
DISTRIBUTION AND ABUNDANCE …............…...……...…... 37
CONCLUSIONS ………………………………………………. 39

PART III: DETERMINING GENETIC RESPONSE TO PRIOR
EXPOSURE AND INFLUENCE ON THE CONCENTRATION
OF PAHS IN THE ENVIRONMENT. ............................................. 41
INTRODUCTION ……………………………………………… 41
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POPULATION DISTRIBUTION AND PAH ATTENUATION............ 45
METHODS................................................................................ 46
14C LABELED PHENANTHRENE STUDY .................................. 46
RESULTS ................................................................................. 48
DISCUSSION ............................................................................ 53

PART IV: SUGGESTIONS FOR FURTHER STUDY ................... 55
TEMPORAL CYCLES AND AROMATIC DIOXYGENASE GENES ............... 55
EFFECTS OF GROUNDCOVER ON DIOXYGENASE GENES ...................... 56
MAPPING MICROBIAL RESPONSE IN A PAH SOURCE/SINK SYSTEM ... 57

LITERATURE CITED ..................................................................... 59

APPENDICES ......................................................................... 66

I: CHATTANOOGA SOILS ...................................................................... 67
II: CNS DATA ...................................................................................... 69
III: RADIORESPIREOMETRY DATA ......................................................... 70
IV: QUANTITATIVE REAL TIME PCR RAW DATA .................................. 72
V: GENE QUANTIFICATION DATA WORKUP.......................................... 80

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LIST OF TABLES

1. PRIMER AND PROBE SEQUENCES FOR NIDA AND NAHAC AS USED IN
THIS STUDY .....................................................................…………… 27

2. WILLIAMS ISLAND DATA BY SAMPLE SITE ……………………….... 30

3. VAAP DATA BY SAMPLE SITE…………………………………....... 32

4. CHATTANOOGA CREEK DATA BY SAMPLE SITE ……………………. 34

5. WILCOXON RANK SUM COMPARISON OF GENES NIDA AND NAHAC
ACROSS THE CHATTANOOGA AREA…................................................ 36

6. CONTAMINANT INFLUENCE ON COMMUNITY STRUCTURE................... 44

7. ENUMERATION DATA SET OF NAHAC AND NIDA AT ALL LOCATIONS .. 48

8. WILCOXON RANK SUM ANALYSIS COMPARING CONCENTRATION OF
NIDA BETWEEN LOCATONS................................................................ 51

9. SPEARMAN CORRELATION BETWEEN NIDA AND 14C PHENANTHRENE
MINERALIZATION IN CHATTANOOGA CREEK ....................................... 52

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LIST OF FIGURES

1. RIESKE GROUP ACTIVE SITE STRUCTURE OF AN AROMATIC
DIOXYGENASE……………………................................................. 8

2. COMPARATIVE CHEMICAL STRUCTURE, THREE COMMON PAHS … 13

3. QUANTITATIVE REAL-TIME PCR RESULTS....................................... 29

4. SHOWING SAMPLING LOCATIONS IN GREATER CHATTANOOGA....... 29

5. SATELLITE IMAGE OF WILLIAMS ISLAND LOCATION..............…….. 30

6. SATELLITE IMAGE OF VAAP LOCATION........................…………… 33

7. SATELLITE IMAGE OF CHATTANOOGA CREEK, COMPARING GENES .. 35

8. RADIORESPIREOMETRY MICROCOSM................................................. 47

9. THE NAHAC GENE IN COPIES/GDRY/CARBON GRAPHED ACROSS
ALL SAMPLE SITES ............................................................................ 48

10. THE NIDA GENE IN COPIES/GDRY/CARBON NIDA GRAPHED ACROSS
ALL SAMPLE SITES............................................................................. 48

11. 14C PHENANTHRENE MINERALIZATION BY CHATTANOOGA CREEK
SOIL SAMPLES IN NGPHENANTHRENE/GDRY/DAY............................. 51

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INTRODUCTION
The field of microbial ecology is advancing rapidly. Recent technological
advances have dramatically expanded the scope and accuracy of scientific techniques for
measuring microbial soil biota. Heretofore, most research in microbial ecology has
emphasized the micro, even the molecular, over the macro. A thorough understanding of
the ecology directly impacting the formation of the bacterial soil environment would be is
necessary for the proper management of soils to human benefit. Thanks to decades of
molecular research, it is now possible for microbial ecologists to understand the
relationship between the microscopic individual bacteria and the systemic
biogeochemical cycles those bacteria drive. In the case of the carbon cycle, human
industrial activity has sequestered carbon in forms actively harmful to human health and
the environment. The biologically driven remediation (bioremediation) of toxic
compounds requires an understanding of the native soil environment and how that
environment responds to human disturbance. Therefore, the primary purpose of this
thesis is to apply quantitative real time PCR and GIS mapmaking technologies to survey
the distribution and concentration of two bacterial aromatic dioxygenase genes associated
with the degradation of a class of recalcitrant, carcinogenic industrial wastes known as
polycyclic aromatic hydrocarbons (PAHs). The secondary purpose is to determine if
these genes do in fact respond to and influence the concentration of PAHs in the
environment.
The physiological ability of certain bacteria to metabolize and degrade PAHs is of
great interest to the scientific community. Bacteria able to completely metabolize and
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